sc(Fv)2s are single-chain antibodies in which two light chain variable regions (VL) and two heavy chain variable regions (VH), four variable regions in total, are linked by linkers or such (Hudson et al., J. Immunol. Methods (1999) 231: 177-189).
For example, single-chain antibodies having the sequence VH1-linker-VL2-linker-VH3-linker-VL4 or VL2-linker-VH1-linker-VL4-linker-VH3 are known. Depending on the combination of Fv (a molecule in which VH and VL are non-covalently linked), two types of structural isomers of sc(Fv)2 would exist: sc(Fv)2 in which each set of Fv is formed by VH1 and VL2, and VH3 and VL4; and sc(Fv)2 in which each set of Fv is formed by VH1 and VL4, and VH3 and VL2.
However, since most previous studies on sc(Fv)2 dealt with bispecific sc(Fv)2s, to date there are almost no reports on structural isomers of sc(Fv)2.
Bispecific sc(Fv)2s are sc(Fv)2s in which the variable regions of VH1 and VL4, and VH3 and VL2 (or VH1 and VL2, and VL3 and VL4) in the VH1-linker-VL2-linker-VH3-linker-VL4 sequence derive from different monoclonal antibodies. In bispecific sc(Fv)2s, VH1 and VL4, or VH3 and VL2 (or VH1 and VL2, or VH3 and VL4) derive from an identical monoclonal antibody. In this case, the efficiency of Fv formation would be higher and therefore the occurrence of structural isomers is suppressed to some extent. In fact, the activity was reported to remain unchanged between bispecific sc(Fv)2s prepared using linkers whose lengths were 15-5-15 and 15-15-15 (Non-patent Document 5). Thus, there is a lack of detailed information regarding structural isomers of sc(Fv)2. For example, Non-patent Documents 3, 4, 8, and 9 indicate the existence of correct Fv combinations confirmed by measuring bispecific binding activities; however, neither a quantitative evaluation regarding the abundance of incorrect Fv combinations nor abundance ratio between the two has been described. Meanwhile, Non-patent Document 6 demonstrates that structural transition between the monomer and the dimer occurs by alteration of lengths of bispecific sc(Fv)2 linkers (alteration of the lengths of linkers at the two ends or in the middle). However, when it comes to structural isomers of sc(Fv)2, the document does not go beyond a discussion on a model-based molecular structure prediction, and describes neither the abundance ratio of the structural isomers nor structural identification in actual samples.
Furthermore, since no attention was focused on structural isomers of sc(FV)2, no close examination on regulating structural isomers was conducted. Non-patent Document 10 also predicts that structures of single chain diabody and bivalent scFv are formed when the length of the linkers are 5-15-5 and 15-5-15, respectively. This is because it has been generally reported in scFvs that adjacent VH and VL are unlikely to form an Fv (i.e., a monomer) when the length of the linker is 12 or shorter. However, Non-patent Document 2 reports that a small quantity of monomers is formed even when the length of the linker in the Fv is 10 or 5. Thus, in the case of Non-patent Document 10, where the linker length is 5-15-5 or 15-5-15, the obtained sc(Fv)2s are not always all in the structural form of single chain diabody or bivalent scFv.
Previous reports evaluated structural isomers by structural prediction based solely upon Fv combinations and linker length. No quantitative analysis of the structural isomer content ratio was conducted. In addition, the obtained structure was not confirmed/verified to see if it was the objective structure. Thus, structural isomers were neither evaluated nor regulated in a sufficient manner. Specifically, regardless of the length, the abundance ratio of the structural isomers of sc(Fv)2 is extremely difficult to predict based on Fv combinations and linker length. The presence of two types of structural isomers is a issue that has to be taken into consideration when sc(Fv)2 molecules comprise two pairs of VH and VL.
There are many known separation methods for optical isomers and geometric isomers of minibody compounds. However, to date there are no reported methods for separating protein isomers. Many methods for separating single amino acid variations in proteins have been previously reported; however, to date, no reports on methods for separating two structural isomers comprising a completely identical amino acid primary sequence is known. The same is true for structural isomers of sc(Fv)2s, and thus, no methods for separating and analyzing, or confirming the two types of structural isomers of sc(Fv)2 existed in prior art.
Since no method was available for separating structural isomers of sc(Fv)2, there are no reports focusing on difference in activity between the two types of structural isomers. In bispecific sc(Fv)2, the activity is obviously predicted to be significantly different between the correct and incorrect Fv combinations within the structural isomers. It is however difficult to predict activity differences between the structural isomers of monospecific sc(Fv)2s that are divalent as well. Non-patent Document 10 ignores the potential differences in activity between the two structural isomers and measures activity (binding activity) using a mixture of the structural isomers. This is because the activity between each structural isomer of sc(Fv)2 could not be strictly compared since highly purified structural isomers could not be prepared because of the difficulty in separation and purification of sc(Fv)2 structural isomers.
Even for sc(Fv)2s with altered linker length, until now it has also been impossible to “identify” (rather than “predict”) each of the two types of structural isomers presumed from linker length and to quantitatively evaluate the content ratio of the structural isomers. Thus, to date, no quantitative evaluation has been performed to reveal the relationship between linker length and content ratio of the structural isomers in sc(Fv)2. Therefore, there are substantially no reports describing the regulation of content ratio of structural isomers by altering linker length.
Alteration of linker length results in the alternation of the distance between the two antigen-binding sites in sc(Fv)2, and thus, linker length has a possible influence on biological activity (agonistic activity such as receptor dimerization). It is thus preferable that the distance between the two antigen-binding sites be arbitrarily adjusted by the lengths of the linkers depending on the type of antigen. Furthermore, linker length has been reported to have a great influence on stability (Non-patent Documents 1 and 2) and the stability of scFvs is known to generally decrease as linkers get shorter. The same would be true for sc(Fv)2s. It is reported that dimers are easily formed by shortening the middle linker (Non-patent Document 6). For the preparation of a highly stable sc(Fv)2, linker lengths that can be arbitrarily adjustable are preferred. When sc(Fv)2s are developed as pharmaceuticals, it is thus preferable that target structural isomers be isolated by their arbitrary linker lengths. However, there are no previous reports describing the isolation of each of the two types of structural isomers, bivalent scFv and single chain diabody, from sc(Fv)2s with linkers of arbitrary lengths.
To develop sc(Fv)2s comprising structural isomers as pharmaceuticals, it is necessary to separate and purify only the targeted structural isomer and to manufacture a bulk drug which comprises only one of the structural isomers. Alternatively; when such a bulk drug is a mixture of structural isomers, it is required to determine the properties of the two types of structural isomers and to conduct a specification test to quantitatively analyze the content ratio of the respective structural isomers. However, to date, there are no known methods for separating and purifying, quantitatively analyzing, or identifying the structural isomers of sc(Fv)2s.
Meanwhile, some reports describe methods for controlling the abundance ratio of monomer/dimer/trimer/tetramer of scFv based on linker length. However, to date, there are no reports describing methods for controlling the abundance ratio of structural isomers by altering linker length because no methods for quantitatively analyzing the structural isomers of sc(Fv)2 have been discovered, as described above.    Non-patent Document 1: Protein Engineering, 1993, 6(8), 989-995    Non-patent Document 2: Protein Engineering, 1994, 7(8), 1027-1033    Non-patent Document 3: Journal of Immunology, 1994, 152, 5368-5374    Non-patent Document 4: Journal of Immunology, 1995, 154, 4576-4582    Non-patent Document 5: PNAS, 1995, 92, 7021-7025    Non-patent Document 6: Journal of Molecular Biology, 1999, 293, 41-56    Non-patent Document 7: Protein Engineering, 2001, 14(10), 815-823    Non-patent Document 8: Journal of Molecular Biology, 2003, 330, 99-111    Non-patent Document 9: Protein Eng Des Sel. 2004 April, 17(4), 357-66    Non-patent Document 10: Clinical Cancer Research, 2004, 10, 1274-1281    Non-patent Document 11: Int. J. Cancer, 1998, 77, 763-772